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Laser contactless ice nucleation for stem cell cryopreservation
Abstracts / Cryobiology 73 (2016) 399e443
generating a “bottle neck” for cellular therapies as well as tissue and organ preservation. The Ben laboratory has discovered several classes of ice recrystallization inhibitors that control ice growth during cryopreservation and increase the post-thaw viability and functionality of human red blood cells, hematopoietic stem cells from umbilical cord blood, and hepatocytes. These compounds have also proven effective in primary endothelial cells where they mitigate cellular damage from intracellular ice recrystallization during cryopreservation at high subzero temperatures. This finding is the first critical step towards using these compounds for the successful cryopreservation of complex tissues and perhaps organs. S075 AVOIDING THE ICE CRYOLESION IN TISSUE CRYOPRESERVATION WITH ICE MODULATORS M. Taylor 1,2, 3, *, Z. Chen 1, C. Crossley 1, E. Greene 1, L. Campbell 1, K. Brockbank 1, Y. Rabin 3. 1 Tissue Testing Technologies LLC, United States; 2 Sylvatica Biotech Inc., Charleston, South Carolina, United States; 3 Carnegie Mellon University, Pittsburgh, Pennsylvania, United States * Corresponding author. Tissue Testing Technologies LLC, United States.
Effective methods of cryopreservation that maintain the function and structural integrity of integrated tissues will not only be realized by effectively controlling ice formation, but must also avoid structural damage due to thermo-mechanical stresses. Building upon published prior work this study focused on defining the structural and functional profile of blood vessels vitrified under marginal conditions of cooling and warming as a function of the system volume and cryoprotectant solution design. A cryomacroscope (Type III) was used to vitrify carotid artery segments in DP6 solutions containing a range of synthetic ice modulators (SIMs) using previously determined marginal thermal conditions for vitrification. The application of SIMs enables lowering the CPA concentration, thereby reducing the toxicity potential, while decreasing the critical cooling and rewarming rates, thereby reducing the risk of structural destruction to the tissue as a consequence of thermo-mechanical stress. Using our established baseline for respectable recovery of function of blood vessels after vitrification in small volumes (1 ml), we determine the outcome of scaleup by orders of magnitude as a crucial step towards successful vitreous cryopreservation of bulky tissues and organs. The impact of scaling up by 400 and 1000% was assessed using glass and plastic specimen vials. At 400% scaling in glass vials > 70% metabolic recovery was achieved for carotid artery segments by the alamarBlue assay and >50% by smooth muscle physiology (contractility). At 1000% it was demonstrated that material properties of the glasses formed result in fracturing at this volume that correlates with losses in functional recovery. Fracturing, prevalent when samples were vitrified in glass containers was avoided in plastic containers with concomitant retention of tissue function. This emphasizes that material properties of the packaging as well as the nature of the vitrification solution impacts the risks of thermo-mechanical stresses and functional recovery of the product. Conflict of interest: All authors except Dr. Yoed Rabin are either full or part-time employees of T3-Tissue Testing Technologies, LLC. Source of funding: This work was supported in part by The Department of Defense DHP-H151-013-0162 SBIR Phase I and NIH, NHLBI, R01HL127618. S076 LASER CONTACTLESS CRYOPRESERVATION
ICE
NUCLEATION
FOR
STEM
CELL
L. Lauterboeck 1, *, W. Wieprecht 2, S. Kabelac 1, B. Glasmacher 1. 1 Leibniz University Hannover, Institute for Multiphase Processes, Hannover, Germany; 2 University of Technology Cottbus, Atmospheric Chemistry and Air Quality, Cottbus, Germany * Corresponding author.
Induced nucleation is used in freezing cells in reproductive medicine and transfusion medicine and has been applied to sperm, oocytes, stem cells, and red blood cells. However, all current induction methods need a direct contact with the sample, such as crystalline cholesterol or sample
419
container, such as cryovials. These methods have the potential to compromise the sample through contamination. Thus, contactless methods such as ultrasound or laser induced nucleation offer high potential. A device was developed that uses a highly focused, 532 nm, nanosecond pulsed laser beam that can induce nucleation at the desired temperature in cryovials. The challenge in this project was to induce nucleation inside a standard cryovial without damaging the vial or cells. The device was validated by comparing electro-freezing, seeding, and laser induced nucleation in multipotent stromal cells (MSCs) of the common marmoset monkey from two different origins (amnion and bone marrow) by determining efficiency of re-cultivation and metabolic activity. No negative effect on cell survival was found when cells were exposed to laser light up to 48 hours. At all tested nucleation temperatures between -4 C and -14 C the amount of successfully induced samples was higher for laser induced nucleation as compared to electro-freezing induction. MSCs had the highest outcome after freezing when the samples were nucleated at -10 C using 5% dimethyl sulfoxide and a two-step freezing protocol (7.5 C/min to -30 C followed by 3 C/min to -80 C). This was true for all three applied nucleation methods and for both analysed stem cell types. To sum up, a contactless method for ice nucleation using green laser light does not harm the stem cells and leads to high cryopreservation outcome. S077 ARTIFICIAL HYPOXIA INDUCIBLE FACTOR-1a (HIF-1a) STABILIZATION CAN ENHANCE CELL AND TISSUE CONSTRUCT SURVIVAL W. Ho*, B. Fuller, G. Jell. University College London, London, United Kingdom * Corresponding author.
Stem cell therapy is a promising technology that has the potential to revolutionise modern medicine. There are, however, challenges associated with cell survival and phenotype stability during cryopreservation, transportation and following implantation. Strategies to increase cell survival from resuscitation and after implantation would enhance the translation potential of cell therapy. Current research has demonstrated that placing cells in mild hypoxia insult can enhance cellular adaptation to adverse environments. The success of hypoxia preconditioning is, however, curtailed by the lack of oxygen on cell growth, phenotype stability, and reoxygenation injury. Cell survival may be artificially increased by stabilising HIF-1a through the use of HIF mimetics, in normoxic, prior to implantation or storage. Stabilising HIF-1a in normoxia may activate several pro-survival factors whilst preventing the oxidative stress associated with hypoxia-reoxygenation injury. In a model of adverse conditions (Saltron perfusion fluid at 4 C for 24 hours), we demonstrated that HepG2 (liver cancerous cells) had increased survival and a higher proliferation rate following resuscitation when exposed to HIF mimetics during cold storage (100 mM Co ions and 250 mM DMOG). Following resuscitation, the cells exposed to the HIF mimetics in normoxia had increased survival (P ¼ <0.01 for Co ions and P ¼ <0.01 for DMOG) and increased proliferation (as determined by total DNA) up to 7 days, (P ¼ <0.05). A similar result was observed in ADMSCs (Adipose Mesenchyme Stem Cell) when incubated in the same dosage of hypoxia mimetics. These results demonstrate that hypoxia mimetics increased the cell survival in adverse conditions e.g. cold storage at 4 C. Current studies are underway to understand the mechanism of this increased survival and resuscitation pro-survival factors expressed in HIF stabilised cells, including the role of ROS and Heat Shock Proteins. The incorporation of HIF mimetics into tissue scaffolds may provide a means to increase cell survival following implantation. S078 TRANSCRIPTOMIC PROFILING REVEALED THE REGULATORY MECHANISM OF ARABIDOPSIS RESPONSE TO OXIDATIVE STRESS FROM CRYOPRESERVATION X. Shen. Shanghai Jiao Tong University, School of Agriculture and Biology, Minhang, Shanghai, China The 48-h Arabidopsis seedlings after germination could be restored, whereas 72-h seedlings died after cryopreservation. To understand the mechanism of this correlation between the seedling age and survival,
generating a “bottle neck” for cellular therapies as well as tissue and organ preservation. The Ben laboratory has discovered several classes of ice recrystallization inhibitors that control ice growth during cryopreservation and increase the post-thaw viability and functionality of human red blood cells, hematopoietic stem cells from umbilical cord blood, and hepatocytes. These compounds have also proven effective in primary endothelial cells where they mitigate cellular damage from intracellular ice recrystallization during cryopreservation at high subzero temperatures. This finding is the first critical step towards using these compounds for the successful cryopreservation of complex tissues and perhaps organs. S075 AVOIDING THE ICE CRYOLESION IN TISSUE CRYOPRESERVATION WITH ICE MODULATORS M. Taylor 1,2, 3, *, Z. Chen 1, C. Crossley 1, E. Greene 1, L. Campbell 1, K. Brockbank 1, Y. Rabin 3. 1 Tissue Testing Technologies LLC, United States; 2 Sylvatica Biotech Inc., Charleston, South Carolina, United States; 3 Carnegie Mellon University, Pittsburgh, Pennsylvania, United States * Corresponding author. Tissue Testing Technologies LLC, United States.
Effective methods of cryopreservation that maintain the function and structural integrity of integrated tissues will not only be realized by effectively controlling ice formation, but must also avoid structural damage due to thermo-mechanical stresses. Building upon published prior work this study focused on defining the structural and functional profile of blood vessels vitrified under marginal conditions of cooling and warming as a function of the system volume and cryoprotectant solution design. A cryomacroscope (Type III) was used to vitrify carotid artery segments in DP6 solutions containing a range of synthetic ice modulators (SIMs) using previously determined marginal thermal conditions for vitrification. The application of SIMs enables lowering the CPA concentration, thereby reducing the toxicity potential, while decreasing the critical cooling and rewarming rates, thereby reducing the risk of structural destruction to the tissue as a consequence of thermo-mechanical stress. Using our established baseline for respectable recovery of function of blood vessels after vitrification in small volumes (1 ml), we determine the outcome of scaleup by orders of magnitude as a crucial step towards successful vitreous cryopreservation of bulky tissues and organs. The impact of scaling up by 400 and 1000% was assessed using glass and plastic specimen vials. At 400% scaling in glass vials > 70% metabolic recovery was achieved for carotid artery segments by the alamarBlue assay and >50% by smooth muscle physiology (contractility). At 1000% it was demonstrated that material properties of the glasses formed result in fracturing at this volume that correlates with losses in functional recovery. Fracturing, prevalent when samples were vitrified in glass containers was avoided in plastic containers with concomitant retention of tissue function. This emphasizes that material properties of the packaging as well as the nature of the vitrification solution impacts the risks of thermo-mechanical stresses and functional recovery of the product. Conflict of interest: All authors except Dr. Yoed Rabin are either full or part-time employees of T3-Tissue Testing Technologies, LLC. Source of funding: This work was supported in part by The Department of Defense DHP-H151-013-0162 SBIR Phase I and NIH, NHLBI, R01HL127618. S076 LASER CONTACTLESS CRYOPRESERVATION
ICE
NUCLEATION
FOR
STEM
CELL
L. Lauterboeck 1, *, W. Wieprecht 2, S. Kabelac 1, B. Glasmacher 1. 1 Leibniz University Hannover, Institute for Multiphase Processes, Hannover, Germany; 2 University of Technology Cottbus, Atmospheric Chemistry and Air Quality, Cottbus, Germany * Corresponding author.
Induced nucleation is used in freezing cells in reproductive medicine and transfusion medicine and has been applied to sperm, oocytes, stem cells, and red blood cells. However, all current induction methods need a direct contact with the sample, such as crystalline cholesterol or sample
419
container, such as cryovials. These methods have the potential to compromise the sample through contamination. Thus, contactless methods such as ultrasound or laser induced nucleation offer high potential. A device was developed that uses a highly focused, 532 nm, nanosecond pulsed laser beam that can induce nucleation at the desired temperature in cryovials. The challenge in this project was to induce nucleation inside a standard cryovial without damaging the vial or cells. The device was validated by comparing electro-freezing, seeding, and laser induced nucleation in multipotent stromal cells (MSCs) of the common marmoset monkey from two different origins (amnion and bone marrow) by determining efficiency of re-cultivation and metabolic activity. No negative effect on cell survival was found when cells were exposed to laser light up to 48 hours. At all tested nucleation temperatures between -4 C and -14 C the amount of successfully induced samples was higher for laser induced nucleation as compared to electro-freezing induction. MSCs had the highest outcome after freezing when the samples were nucleated at -10 C using 5% dimethyl sulfoxide and a two-step freezing protocol (7.5 C/min to -30 C followed by 3 C/min to -80 C). This was true for all three applied nucleation methods and for both analysed stem cell types. To sum up, a contactless method for ice nucleation using green laser light does not harm the stem cells and leads to high cryopreservation outcome. S077 ARTIFICIAL HYPOXIA INDUCIBLE FACTOR-1a (HIF-1a) STABILIZATION CAN ENHANCE CELL AND TISSUE CONSTRUCT SURVIVAL W. Ho*, B. Fuller, G. Jell. University College London, London, United Kingdom * Corresponding author.
Stem cell therapy is a promising technology that has the potential to revolutionise modern medicine. There are, however, challenges associated with cell survival and phenotype stability during cryopreservation, transportation and following implantation. Strategies to increase cell survival from resuscitation and after implantation would enhance the translation potential of cell therapy. Current research has demonstrated that placing cells in mild hypoxia insult can enhance cellular adaptation to adverse environments. The success of hypoxia preconditioning is, however, curtailed by the lack of oxygen on cell growth, phenotype stability, and reoxygenation injury. Cell survival may be artificially increased by stabilising HIF-1a through the use of HIF mimetics, in normoxic, prior to implantation or storage. Stabilising HIF-1a in normoxia may activate several pro-survival factors whilst preventing the oxidative stress associated with hypoxia-reoxygenation injury. In a model of adverse conditions (Saltron perfusion fluid at 4 C for 24 hours), we demonstrated that HepG2 (liver cancerous cells) had increased survival and a higher proliferation rate following resuscitation when exposed to HIF mimetics during cold storage (100 mM Co ions and 250 mM DMOG). Following resuscitation, the cells exposed to the HIF mimetics in normoxia had increased survival (P ¼ <0.01 for Co ions and P ¼ <0.01 for DMOG) and increased proliferation (as determined by total DNA) up to 7 days, (P ¼ <0.05). A similar result was observed in ADMSCs (Adipose Mesenchyme Stem Cell) when incubated in the same dosage of hypoxia mimetics. These results demonstrate that hypoxia mimetics increased the cell survival in adverse conditions e.g. cold storage at 4 C. Current studies are underway to understand the mechanism of this increased survival and resuscitation pro-survival factors expressed in HIF stabilised cells, including the role of ROS and Heat Shock Proteins. The incorporation of HIF mimetics into tissue scaffolds may provide a means to increase cell survival following implantation. S078 TRANSCRIPTOMIC PROFILING REVEALED THE REGULATORY MECHANISM OF ARABIDOPSIS RESPONSE TO OXIDATIVE STRESS FROM CRYOPRESERVATION X. Shen. Shanghai Jiao Tong University, School of Agriculture and Biology, Minhang, Shanghai, China The 48-h Arabidopsis seedlings after germination could be restored, whereas 72-h seedlings died after cryopreservation. To understand the mechanism of this correlation between the seedling age and survival,